Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Avermectin, candicidin, epothilone, erythromycin, FK-506, FK-520, narbomycin, oleandomycin, picromycin, rapamycin, spincoyn, tetracycline, and tylosin are examples of such compounds. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low expression of polyketides in wild-type cells that produce them naturally, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.
This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. For example, the following publications relate generally to the cloning of all or parts of the genes coding for the expression of PKS enzymes or other enzymes that act on polyketides of significant commercial interest or potential.
Avermectin
U.S. Pat. No. 5,252,474 to Merck.
MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.
MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.
Candicidin (FRO008)
Hu et al., 1994, Mol. Microbiol. 14: 163-172.
Epothilone
U.S. patent application Ser. No. 60/130,560, filed Apr. 22, 1999, and Ser. No. 60/122,620, filed Mar. 3, 1999.
Erythromycin
PCT Pub. No. 93/13663 to Abbott.
U.S. Pat. No. 5,824,513 to Abbott.
Donadio et al., 1991, Science 252:675-9.
Cortes et al., Nov. 8, 1990, Nature 348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea.
Glycosylation Enzymes
PCT Pat. App. Pub. No. 97/23630 to Abbott.
FK-506
Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506, Eur. J. biochem. 256: 528-534.
Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506, Eur. J. Biochem. 244: 74-80.
Methyltransferase
U.S. Pat. No. 5,264,355, issued Nov. 23, 1993, Methylating enzyme from Streptomyces MA6858. 31-O-desmethyl-FK506 methyltransferase.
Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase 40 genes involved in the biosynthesis of the immunosuppressants FK506 and FK520, J. Bacteriol. 178: 5243-5248.
FK-520
U.S. patent application Ser. No. 60/139,650, filed Jun. 17, 1999, and Ser. No. 60/123,810, filed Mar. 11, 1999. See also Nielsen et al., 1991, Biochem. 30:5789-96 (enzymology of pipecolate incorporation).
Lovastatin
U.S. Pat. No. 5,744,350 to Merck.
Nemadectin
MacNeil et al., 1993, supra.
Niddamycin
Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis, J. Bacteriol. 179: 7515-7522.
Oleandomycin
Swan et al., 1994, Characterisation of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Mol. Gen. Genet. 242: 358-362.
U.S. patent application Ser. No. 60/120,254, filed Feb. 16, 1999.
Olano et al., 1998, Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308.
Platenolide
EP Pat. App. Pub. No. 791,656 to Lilly.
Rapamycin
Schwecke et al., August 1995, The biosynthetic gene cluster for the polyketide rapamycin, Proc. Natl. Acad. Sci. USA 92:7839-7843.
Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.
Rifamycin
August et al., Feb. 13, 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry & Biology, 5(2): 69-79.
Soraphen
U.S. Pat. No. 5,716,849 to Novartis.
Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangium cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.
Spiramycin
U.S. Pat. No. 5,098,837 to Lilly.
Activator Gene
U.S. Pat. No. 5,514,544 to Lilly.
Tylosin
EP Pub. No. 791,655 to Lilly.
Kuhstoss et al., 1996, Gene 183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.
U.S. Pat. No. 5,876,991 to Lilly.
Tailoring enzymes
Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355. Analysis of five tylosin biosynthetic genes from the tylBA region of the Streptomyces fradiae genome.
Each of the above-referenced patent applications, patents, and publications is incorporated by reference herein.
The cloning of PKS genes has been accompanied by advances in technology allowing one to manipulate a known PKS gene(s) either to produce the polyketide synthesized by the corresponding PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketide produced from a known PKS. See, e.g., PCT publication Nos. WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 5,672,491; and 5,712,146; and Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Cane et al., Oct. 2, 1998, Harnessing the Biosynthetic Code: Combinations, Permutations, and Mutations, Science 282: 63-68, each of which is incorporated herein by reference.
PKS enyzmes are similar to, but distinct from, the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. Two major types of PKS enzymes are found in nature: these types are commonly referred to as Type I or "modular" and Type II "aromatic" PKS enzymes. A third type sometimes referred to in the scientific literature is a "fungal PKS"; however, for purposes of the present invention, this type is to be considered a Type I PKS. These types differ in their composition and mode of synthesis of the polyketide synthesized. Type I PKSs are typically found in nature as complexes of multiple very large proteins. In this type, a set of separate catalytic active sites (each active site is termed a "domain", and a set thereof is termed a "module") exists for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway.
The active sites and modules of a typical Type I PKS enzyme are shown in FIG. 9 of PCT patent publication No. WO 95/08548, which depicts a model of 6-deoxyerythronolide B synthase ("DEBS"), which is involved in the synthesis of erythromycin. Six separate modules, each catalyzing a round of condensation and modification of a 2-carbon unit, are present in DEBS. The number and type of catalytic domains that are present in each module varies, and the total of 6 extender modules and a loading module is provided on 3 separate proteins (designated DEBS-1, DEBS-2, and DEBS-3, with 2 modules per protein). The catalytic domains of the DEBS polypeptides provide a representative example of Type I PKS structure. In this particular case, the loading module and extender modules 1 and 2 reside on DEBS-1, extender modules 3 and 4 on DEBS-2, and extender modules 5 and 6 on DEBS-3; module 1 is the first module to act on the growing polyketide backbone, and module 6 the last. Each module of consists of at least two (if a loading module) and more typically three or more enzymatic activities or "domains."
A typical (non-starter) minimal Type I PKS module is typified by module 3 of DEBS, which contains a ketosynthase ("KS") domain, an acyltransferase ("AT") domain, and an acyl carrier protein ("ACP") domain. These three enzyme activities are sufficient to activate the 2-carbon extender unit and attach it to the growing polyketide molecule. Additional domains that may be included in a module relate to reactions other than the actual condensation, and include a ketoreductase activity ("KR") activity, a dehydratase activity ("DH"), and an enoylreductase activity ("ER"). With respect to DEBS-1, the first module thereof also contains repeats of the AT and ACP activities because it catalyzes initial condensation, i.e., it begins with a "loading domain" consisting of an AT and an ACP domain that determines the nature of the starter unit.
The "finishing" of the 6-deoxyerythronolide molecule is regulated by a thioesterase ("TE") activity in module 6. The TE activity catalyzes cyclization of the macrolide ring by formation of an ester linkage. In FK-506, FK-520, rapamycin, and similar polyketides, the ester linkage formed by the TE activity is replaced by a linkage formed by incorporation of a picolate acid residue. The enzymatic activity that catalyzes this incorporation for the rapamycin enzyme is known as rapP.
In PKS polypeptides, the regions that confer enzymatic activity (domains) are separated by linker or "scaffold"-encoding regions. These scaffold regions encode amino acid sequences that space the enzymatic activities (domains) at the appropriate distances and in the correct order. Thus, the linker regions of a PKS protein collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the DNA level) between PKS enzymes by various available methodologies. Thus, there is considerable flexibility in the design of new PKS enzymes with the result that known polyketides can be produced more effectively, and novel polyketides useful as pharmaceuticals or for other purposes can be made.
Type I PKS enzymes are encoded by "PKS gene clusters." PKS gene clusters usually consist of three or more open reading frames ("ORFs"), each encoding two or more modules of ketosynthase activity. For example, each of the DEBS polypeptides is encoded by a separate open reading frame (ORF). See Caffrey et al., 1992, FEBS Letters 304: 205, incorporated herein by reference.
As noted above in connection with reference to enzymes that cyclize linear polyketides, additional structural complexity in polyketides arises from or can be introduced by various activities, including glycosylation, hydroxylation, methylation, and other enzymatic activities. The rapP enzymatic activity mentioned above is an example of one such activity; another example is the hydroxylation of a polyketide by an oxidase enzyme similar in structure and function to the cytochrome P450 oxidase enzyme. The genes encoding such enzymatic activities are often found in relatively close proximity to the PKS genes and so may be considered part of a PKS gene cluster. By appropriate application of recombinant DNA technology, a wide variety of polyketides can be prepared in a variety of different host cells provided one has access to nucleic acid compounds that encode PKS proteins and polyketide modification enzymes.
The present invention helps meet the need for such nucleic acid compounds, recombinant PKS enzymes, and recombinant enzymes that modify polyketides by providing recombinant vectors that encode the narbonolide PKS enzyme and various narbomycin modification enzymes.